HIV-infected individuals who abuse heroin are at greater risk for HAND. The synaptodendritic injury that underlies HAND is largely mediated by infected and/or activated microglia and astroglia as neurons are rarely, if ever, infected. CNS neurons and glia are derived from neuroepithelial precursors in the subventricular zone (SVZ). In development, undifferentiated, common neural progenitor cells (NPCs) give rise to cells that form neurons, astroglia, and oligodendroglia on a precise schedule of proliferation, migration, and differentiation regulated by complex intra- and extracellular signals. In the mature CNS, neurogenesis is largely limited to specific regions (dentate gyrus and SVZ of lateral ventricles), while gliogenesis also occurs in the parenchyma. During the 1st funding period, we showed that HIV-1 and HIV proteins diminished NPC ability to differentiate and populate the striatum, in mice exposed both perinatally and as adults, and in vitro. In general, HIV R5 (culture) and HIV-1 Tat (in vivo stereology; culture) reduced Sox2+ NPCs and slowed proliferation. HIV-1 Tat in vivo also seemed to redirect cell fate as mature cell populations were changed ( oligodendroglia, astroglia) while total cell numbers and volume were normal. Co-exposure to morphine exacerbated some outcomes. Thus, both HIV and Tat interfere with NPC development via mechanisms that opiates can amplify. Data demonstrate that R5 or chronic Tat exposure distort normal NPC microglia interactions, resulting in abnormal CNS populations. Imbalances ( inflammatory astroglia, myelinating OLs) likely contribute to HIV-related deficits in CNS function. We hypothesize that HIV by itself, and more potently with morphine, overactivate microglia, causing aberrant signaling to NPCs through NADPH oxidase (NOX2) and iNOS/nitric oxide. The inappropriate signaling misdirects NPC lineage/differentiation, ultimately altering CNS cell populations. This theory is explored in vivo (mouse/HIV-1 Tat model) and in human brain cultures (HIV infective model) in 2 related aims. Aim 1 tests if microglia play a central rol in the aberrant proliferation/lineage of NPCs after exposure to HIV-1 Tat opiates. Studies use a macrophage/ microglia deficient transgenic model (HIV-1 Tat x CSF1op/op mice), with chronic Tat induction at both adolescent (high risk for HIV and opiate exposure through exploratory behaviors) and adult stages. Hippocampus and striatum are studied; motor and cognitive behaviors correlated to population changes. NPCs are also examined in HIV+/HIV- autopsy tissue. Aim 2 tests the roles of microglial NOX2 and iNOS signaling as causal in dysregulated NPC dynamics, using 3-dimensional aggregate cultures from human brain and real-time tracking of individual human NPCsHIVopiatesspecific inhibitors of iNOS, NOX2 and R5 binding. Amnis flow-single cell imaging used to identify infected cells.